1. Field of the Invention
The present invention relates to methods and agents useful for suppressing NF-κB activation. The present invention also relates to methods and agents useful for treating and/or preventing inflammation. The present invention further relates to methods and agents useful for the treatment or prophylaxis of diseases caused by NF-κB activation.
2. Discussion of the Background
Inflammation is a biological response to various exogenous or endogenous tissue-damaging stimuli. When a part of the body tissue is damaged by infection and the like, various biological response-modifying substances are produced in and released from the tissue, and cause inflammation. In the initial stages, inflammatory cytokines such as TNF-α, IL-1 and the like are produced in the inflammation site, expression of cell adhesion factors (ICAM-1, VCAM-1 etc.) on the vascular endothelium is enhanced by the action of the cytokines, and adhesion of the inflammatory cells to the vascular wall is promoted. Simultaneously, monocytes, lymphocytes and neutrophils infiltrate into the inflammation tissue due to the chemotactic factors produced in the inflammation site, such as MCP-1, IL-8 and the like. While various cytokines, active oxygen, hydrolase and the like are additionally secreted from the subendothelially infiltrated inflammatory cells against pathogenic microorganisms and the like, they also damage the host tissue at the same time. When the proinflammatory substance is eradicated or removed through such processes, infiltration of the inflammatory cell generally stops and the inflammation progresses towards dissipation. When the tissue damage is high, however, it is not repaired completely, and the parenchymal cell is replaced (fibrosed) by connective tissues, sometimes causing dysfunction of the tissue. In addition, continuous infection such as tuberculosis, self-tissue damage due to autoimmune mechanism, accumulation of oxidized LDL and the like prolong inflammation and sometimes form pathology of repeated active inflammatory response, tissue destruction and tissue repair over a long period, which is called chronic inflammation. Quite a number of such chronic inflammatory diseases are intractable, such as Behcet's disease and the like, and patients suffer from decreased quality of life (QOL) for a long time.
In these inflammation processes, many genes are activated, and NF-κB is a transcription factor playing a key role in the gene activation process. It is known that NF-κB is a heterodimer consisting of two subunits p50 and p65, and usually present in the cytoplasm in an inert form of a complex with I-κB, which is an inhibitory protein. It is known that, when stimulated, however, I-κB is degraded by phosphorylation and released from the complex, then the NF-κB heterodimer is translocated into the nucleus, binds to DNA and regulates the gene expression in the downstream of the binding site. It is said that oxidative stress is involved in the process of activation of NF-κB and translocation into the nucleus. While the mechanism thereof is yet to be clarified in a number of aspects, it is considered that a certain kind of reactive oxygen species (ROS) activates I-κB kinase and promotes degradation of I-κB. Addition of a plurality of antioxidants and overexpression of antioxidant enzymes such as glutathione peroxidase and the like are known to suppress activation of NF-κB. Conversely, binding of activated NF-κB, which is translocated into the nucleus, onto a DNA is inhibited by an oxidant and promoted by thiols. Thus, it can be said that activation of NF-κB and subsequent expression of various gene products are both under the control of redox. However, since the manner of control is different, the influence of the changes in the intracellular redox state on the gene product expression under the control of NF-κB is not always uniform but complicated (see, Halliwell B and Gutteridge J M, Free Radicals in Biology and Medicine Third Edition, Oxford Science Publications (1999)). Representative examples of the gene products subject to expression control by NF-κB include inflammatory cytokines such as TNF-α, IL-1β, IL-6 and the like; cell adhesion factors such as ICAM-1, VCAM-1, E-selectin and the like; cell chemotactic factors (chemokine) such as IL-8 or MCP-1 and the like; inducible nitric oxide synthase (iNOS); tissue factor (TF); inducible cyclooxygenase (COX-2); and the like. Activation of NF-κB is considered to be involved in many diseases. Examples of important diseases include atherosclerosis, myocardial infarction, virus infection (HIV, cytomegalovirus and the like), arthritis (chronic rheumatoid arthritis, osteoarthritis and the like), psoriasis, inflammatory bowel disease (IBD), type II diabetes, bronchial asthma, sepsis, autoimmune diseases and the like.
VCAM-1 (Vascular Cell Adhesion Molecule-1), which shows increased expression by the activation of NF-κB, is a glycoprotein having a molecular weight of 110 kDa, and the expression thereof is mainly observed in vascular endothelial cells, macrophages and the like. The main action thereof is to strongly adhere leukocytes onto vascular endothelial cells by binding to VLA4, one of the members of the β1-integrin family, which is expressed in lymphocytes, monocytes and the like. As for the relationship between VCAM-1 and diseases, increased expression of VCAM-1 has been confirmed in the vascular endothelial cells of topical lesions of various acute/chronic inflammatory diseases such as atherosclerosis, allograft rejection, metastasis of malignant tumor (melanoma etc.) and the like.
MCP-1 (Monocyte Chemotactic Protein-1) is produced by endothelial cells, smooth muscle cells, macrophages and the like, and strongly induces subendothelial migration of monocyte/macrophage and T lymphocyte adhered to the vascular endothelial cell. Therefore, MCP-1 is considered to promote tissue infiltration of monocyte and T cell at various inflammatory lesions. MCP-1 is also known to have associations with many diseases, and is reported to show enhanced production at topical sites, as well as improvement of symptoms by MCP-1 neutralization antibody/antagonist and the like, in chronic inflammatory diseases such as bronchial asthma, allergic rhinitis, chronic rheumatoid arthritis, lung hypertension, hepatic fibrosis, renal sclerosis, viral encephalitis, atherosclerosis, endometriosis, inflammatory bowel disease (IBD) and the like, and allergic disease/immune abnormality.
While various anti-inflammatory agents are used for treating inflammatory diseases, a decisive agent which suppresses production of various inflammatory mediators or expression of inflammatory cell adhesion molecules has not been found. One of the reasons therefor is that blocking of a single enzyme activity or single cytokine production is considered to be insufficient, since many gene products are involved in inflammatory responses, as mentioned above. For example, NSAIDs (non-steroidal anti-inflammatory drugs) suppress production of inflammatory prostaglandin by inhibiting cyclooxygenase in arachidonic acid metabolism, but they do not directly inhibit cytokine production. Even if an anticytokine therapy using a cytokine antibody or a cytokine receptor blocker and the like can suppress function of a particular cytokine, it cannot directly suppress the activity of plural cytokines. For example, anti-ICAM-1 antibody is reported to be effective for suppressing rejection during organ transplantation and the like, but it does not have a direct action on other adhesion molecules or inflammatory cytokines. In addition, although anti-cytokine therapy shows a high initial administration effect, the duration of the effect is questioned, and clinical application thereof to chronic inflammatory diseases is considered to be difficult. Meanwhile, conventional anti-inflammatory agents containing an immunosuppressant and anti-cytokine therapy reportedly cause many side effects. For example, steroids can be administered orally and advantageously non-specifically suppress production of plural cytokines. On the other hand, it includes the problems of aggravated infection, rebound and the like. Although anti-TNF-α antibody shows an effect on chronic rheumatoid arthritis and Crohn's disease, serious problems of infectious diseases, allergy (anaphylactic shock) and the like have been pointed out.
Since NF-κB suppressive agents inhibit a common process in the activation of a gene involved in the production of the aforementioned various inflammation causative substances, in contrast to the existing anti-inflammatory agents, there is a possibility that such agents may act widely and anti-inflammatorily. In other words, since the agent simultaneously suppresses transcription of a plurality of inflammatory cytokines, it may be applied to a pharmaceutical product for treating diseases caused by abnormal production of inflammatory cytokines, an antiviral agent against viruses requiring activation of NF-κB for self-replication, such as HIV, cytomegalovirus and the like, an anti-inflammatory agent or cancer metastasis suppressant based on the suppression of expression of cell adhesion molecules, an immunosuppressant used in organ transplantation, and the like.
Some natural components contained in food and the like are also known to have an NF-κB suppressive action (quercetin (see, Martinez-Florez S et al., J. Nutr. 135:1359-1365 (2005)), curcumin (see, Yeh C-H et al., J. Surgical Res. 125:109-116 (2005)), isovitexin (see, Lin C-M et al., Planta Medica 71:748-753 (2005)), apigenin (see, Gerritsen M E et al., Am. J. Pathol. 147:278-292 (1995)), pycnogenol (see, Packer L, Book of abstracts, 219th ACS National Meeting, San Francisco, Calif., Mar. 26-30, (2000)), and the like). These polyphenols are taken orally, considered to have comparatively high safety, and some of them are reported to show an antioxidant activity and an anti-atherosclerosis action and the like (see, Nakamura Y et al., Jpn. J. Cancer Res. 89:361-370 (1998) and Karnada C et al., Free Rad. Res. 39:185-194 (2005)). Therefore, they may be useful for daily control of chronic inflammatory condition or prevention of the onset of inflammatory diseases. However, these suppressive actions of NF-κB were examined in vitro, whereas polyphenols are reportedly metabolized in the body to show an attenuated antioxidant action or lose the action (see, Williamson G et al., Free Rad Res. 39:457-469 (2005)). Therefore, it is unknown whether these substances actually show an anti-inflammatory action in the body.
Safflower seed defatted meal extracts are shown to strongly suppress oxidation of low-density lipoprotein (LDL) in vitro, and suppress formation of arteriosclerotic lesion in apoE KO mouse (see, WO2003/086437), as well as improve blood pressure or pulse wave of KHC rabbit, which is an atherosclerosis model, or human volunteers (see, WO2007/032551). The Hydroxycinnamic acid serotoninamide (serotonin derivative) contained in the extract is considered to be at least one of the anti-arteriosclerotic active forms in apoE KO mouse (see, WO2003/086437). As the serotonin derivative in a safflower seed, four kinds of N-(p-Coumaroyl)serotonin (CS), N-Feruroyl serotonin (FS) and glycosides thereof are mainly known, and the structures of these compounds are completely different from that of the NF-κB suppressant shown in the above-mentioned Martinez-Florez S et al., J. Nutr. 135:1359-1365 (2005); Yeh C-H et al., J. Surgical Res. 125:109-116 (2005); Lin C-M et al., Planta Medica 71:748-753 (2005); Gerritsen M E et al., Am. J. Pathol. 147:278-292 (1995); and Packer L, Book of abstracts, 219th ACS National Meeting, San Francisco, Calif., Mar. 26-30, (2000). CS and FS are both shown to have almost the same level of antioxidant activity (DPPH radical scavenging activity, LDL oxidation suppressive action and the like). Of these, CS is reported by Kawashima et al. to suppress production of inflammatory cytokines such as TNF-αc, IL-1, IL-6 and the like in human peripheral blood monocyte stimulated by lipopolysaccharide (LPS) of bacterium (see, Kawashima S et al., J. Interferon Cytokine Res. 18:423-428 (1998)). According to this report, CS inhibited production of TNF-α, IL-1α, IL-1β and IL-6 at a transcription level at a concentration of 50 μM or above, and inhibition of NF-κB activation was confirmed from the results of EMSA (Electrophoretic Mobility Shift Assay). However, in the follow-up report by the same research group (see, Takii T et al., Int. Immunopharmacol. 3:273-277 (2003)), it is observed that the above-mentioned effect of CS is not specific to inflammatory cytokines, but results from a non-specific protein synthesis inhibitory action and an antioxidant action of CS in peripheral blood-derived monocyte. Taking together all these findings, the effect of a serotonin derivative on the expression of inflammatory cytokines or NF-κB activity in a cell other than peripheral blood monocyte is unknown.
Thus, there remains a need for agents and methods for treating inflammation.